Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography

This protocol can be used to measure patterns of breathing in mice. Importantly, it can minimize the impact of active behaviors in genetically modified strains that can confound results. The main advantages of this technique are that it is noninvasive and that it does not require the use of anesthetic or stress-inducing restraints.

Begin by making sure that all of the hoses and tubes are connected to the barometric plethysmograph chamber and by connecting a gas inflow tube and a vacuum outflow tube directly to the barometric plethysmography chamber. To calibrate the low flow into the chamber, set the flow into and out of the chamber to zero and remove the flow tube into the chamber. Turn off the vacuum and enter a zero into the low unit cell to record the zero flow in the 7700 amplifier setup of the barometric plethysmograph software.

Then reattach the inflow tube and turn on vacuum flow to calibrate the high flow and to allow 20.93%oxygen balanced nitrogen gas to flow through the barometric plethysmography chamber from the gas mixer. Next, convert the inflow value measured from the flow meter from liters per minute into milliliters per second and click the high unit cell to enter the value in milliliters per second. Double-click high cal, change the time to three seconds and click Measure.

Then leave the 7700 amplifier setup tab open to calibrate the metabolic analyzers to the barometric plethysmography software. To calibrate the metabolic analyzer, in the gas mixer program, set the gas mixer to 20.93%oxygen and 79.07%nitrogen. And on the metabolic analyzer, set the oxygen calibration level to 20.93%and the carbon dioxide to 0%Turn the dial back to sample once the appropriate values have been entered on the gas analyzers to set the high oxygen and low carbon dioxide percentage.

Click on the ABCD 4 tab of the barometric plethysmography software and enter 20.93 under high unit of the C2 line for oxygen. Under high cal, change the time to three seconds and click Measure. Enter zero under low cal of the C3 line for carbon dioxide.

Then change the time to three seconds and click Measure under low cal. In the gas mixer program, change the oxygen value to 10%and the carbon dioxide value to 5%and wait several minutes for the gas flow to adjust to these values. On the metabolic analyzers, turn the adjustment knobs to calibrate the carbon dioxide to 5%taking care to turn the dial back to sample once the values have been calibrated.

After checking that the analyzer readings are stable, click high unit under C3 and enter five for carbon dioxide. Then change high cal to three seconds and click Measure. Click low unit under the C2 option and enter 10 for oxygen and click low cal, input three seconds and click Measure.

Change the gas mixer back to 20.93%oxygen and 79.07%nitrogen and wait several minutes for the chamber to adjust to these values. Routinely run additional calibrations with certified gas tanks to confirm the flow mixer and oxygen-carbon dioxide analyzers are performing properly. When the metabolic analyzers have been calibrated, recheck the flow meters connected to the barometric plethysmograph chamber and adjust the air flow into and out of the chamber to rates appropriate for the experiment.

When all of the settings have been applied to the barometric plethysmography software, click Close in the Acquisition tab. Hit Start Acquisition, name the file and click OK to begin recording. For unrestrained barometric plethysmography, record the weight and initial body temperature of the mouse before returning the mouse to its home cage.

Wait 10 minutes to collect the background oxygen and carbon dioxide data from the empty chamber before placing the mouse into the chamber. During the first hour, document the behavior of the animal taking detailed notes that include the specific values of the in and outflow of the chamber. At the end of the chamber habituation, watch for segments of quiet breathing for the next 60 minutes taking body temperature measurements every 10 minutes when using an implantable device.

Take care to become familiar with general mouse behaviors so that instances of calm breathing with no sniffing, grooming or exploring can be appropriately identified. At the end of the experiment, return the mouse to its cage and thoroughly clean and wipe down the equipment. For a metabolism analysis, open the metabolic panel in the software and obtain the average of the first 10 minutes of the oxygen and carbon dioxide levels from when the chamber was empty.

View the flow panel of the barometric plethysmography software and right-click Analyze Attribute to set appropriate parameters. For a pattern of breathing analysis, confirm the times for the 15 seconds of quiet breathing using notes about animal behavior as well as the flow panel tracing. Then enter the times under open parser dialogue from the data parser tab and click Parser View Mode to show only the specific 15-second segments of interest.

To analyze apneas and augmented breath, in the open review file, exit the parser view mode and click Setup P3 Setup and Graph Setup to select Page View as the type and five as the number of panes. Enter minus two into the box labeled low and two into the box labeled high for the flow measures in milliliters per second and apply the changes. Then scroll to the 30-minute mark on the flow tracings panel and manually count the apneas and augmented breaths for the 30 to 60-minute period after the mouse was placed in the chamber.

For this analysis, periods of suspended breathing lasting longer than or equal to 0.5 seconds are indicative of an apnea. Augmented breaths are indicated by a sharp rise in the breathing trace above 1.25 milliliters per second followed by a sharp decrease below minus 0.75 milliliters per second. This representative flow tracing of quiet breathing in a 22-month-old mouse is typical of a pattern in which the breathing is consistent with no active breathing behaviors.

This breathing pattern is from a more active segment during which the mice were exploring the chamber sniffing and/or grooming and is not ideal for the type of breathing collection used for this methodology. The parameter selected for the assessment of possible pattern of breathing differences between the two time points were breathing frequency, tidal volume, minute ventilation, tidal volume inspiratory time ratio, and minute ventilation expelled carbon dioxide ratio. Further analysis was performed with each of the four 15-second baseline segments for frequency, tidal volume, minute ventilation, tidal volume inspiratory time ratio, and minute ventilation expelled carbon dioxide ratio.

No significant differences between any of the time points and no differences in the variability between any of the four time segments for any pattern of breathing measure were found. Quantification of the number of apneas and augmented breaths observed for each animal during minutes 30 to 60 of the unrestrained barometric plethysmography protocol revealed that aged animals showcased a high number of apneas and the presence of augmented breaths within a 30-minute span. Researchers can also employ gas challenges such as hypoxia or hypercapnia to document any changes in the breathing pattern resulting from an intervention or therapy.

This technique provides a means for characterizing patterns of breathing in small rodents and genetically modified animal strains that may exhibit distinct behavioral differences.